Collection tubes apparatus, systems, and methods

ABSTRACT

Methods of producing collection tubes are presented. The methods include providing a separator substance that can rapidly polymerize in a short time to a desired hardness and disposing the separator substance within the lumen of the tube. The separator substance is formulated to have a density between an average density of a serum fraction of whole blood and a cell-containing fraction of whole blood, and to be flowable with whole blood. Upon centrifugation of a tube having blood, the separator substance forms a barrier between the whole blood fractions. The barrier rapidly hardens forming a solid barrier when triggered by a suitable energy source.

This application is a divisional of co-owned pending U.S. applicationSer. No. 12/691,569 filed Jan. 21, 2010 which is a divisional ofco-owned, U.S. Pat. No. 7,673,758 issued Mar. 9, 2010 which is acontinuation-in-part of co-owned U.S. Pat. No. 7,674,388 issued Mar. 9,2010 which claims priority to co-owned U.S. provisional patentapplication 60/707,299 filed Aug. 10, 2005. These and all otherextrinsic references are incorporated herein by reference in theirentirety. Where a definition or use of a term in an incorporatedreference is inconsistent or contrary to the definition of that termprovided herein, the definition of that term provided herein applies andthe definition of that term in the reference does not apply.

FIELD OF THE INVENTION

The field of the invention is separation technologies.

BACKGROUND

Analysis of blood samples often requires separation of whole blood intoa serum fraction and a cell-containing fraction. It is well known in theart that whole blood separation can be carried out throughcentrifugation by disposing whole blood into a blood collection tube,placing the tube into a centrifuge, and spinning down the blood.

Unfortunately, once the blood separates, the fractions of the wholeblood can remix causing contamination of the fractions throughdiffusion, agitation, sample extraction, or other undesirableinteraction. Ideally, the two fractions should remain isolated to ensureno contamination occurs when accessing the desired fraction.

Any system that isolates the fractions of whole blood must include aseparator substance having a suitable density within the tube. Suitabledensities are about 1.04 g/cm³ and are between the density of theheavier cell-containing phase and the density of the lighterserum-containing phase. When whole blood is added to the tube and thetube is centrifuged, the separator substance migrates to between thefractions isolating the two fractions from each other. An examplecollection tube using a gel as a separator substance and that isflowable with whole blood can be found in U.S. Pat. No. 4,946,601 toFiehler. An example separator substance that is also flowable with wholeblood can be found in U.S. Pat. No. 6,248,844 and U.S. Pat. No.6,361,700 to Gates et. al. In those patents the substance is a polyestercurable to a desired viscosity.

Although providing a flowable substance allows for separating thefractions of whole blood, flowable substances have severaldisadvantages. A flowable substance remains flowable even aftercentrifugation which results in a risk of contamination of the sample ifproper care is not taken to keep the sample suitably still and protectedfrom agitation. For example, it is known to use a thixotropic gel in ablood collection tube before centrifugation.

U.S. Pat. No. 4,818,418 to Saunders discusses the use of a thixotropicgel in blood collection tubes. The problem with thixotropic gels,however, is they do not form a sufficiently permanent separation barrierbetween the fractions of whole blood. When a sample is extracted fromthe tube with a pipette, the substance can contaminate or plug thepipette if it touches the substance due to the flowable nature of thesubstance. If the substance is formulated or configured with a highviscosity to provide a sufficiently solid or permanent barrier toovercome the previous disadvantages, then the substance is no longersuitably flowable with whole blood resulting in prohibitive centrifugetimes. Short centrifuge times are critical in life or death situationswhere a blood analysis result is required quickly.

An alternative approach taken by collection tube manufactures is toprovide moveable solid barriers. Examples of suitable solid substancesinclude the intermediate density polymers found in U.S. Pat. No.3,647,070 where polymer spheres form the barrier layer. U.S. Pat. No.5,266,199 describes a tube-and-ball valve that controls separation ofthe serum from the cell-containing phase. However, such physicalbarriers do not provide a sufficient seal between the fractions and areoften either incomplete and tend to leak, or impracticable for othervarious reasons.

These and other solutions for whole blood separation lack the necessaryfeatures to ensure the separated factions of whole blood are effectivelyprotected against contamination due to undesirable sample interactionswhile supporting short centrifugation times. Thus, there is still a needfor liquid separation technologies in which the separation layer issolidified after centrifugation.

SUMMARY OF THE INVENTION

The present invention provides apparatus, systems and methods in which aseparation layer is solidified after centrifugation.

In preferred embodiments the separation layer is hardened throughpolymerization. Suitable separator substances are formulated to have asuitable density intermediate to the fractions of the liquid beingseparated. Where the liquid being separated is whole blood, for example,the separator substance would be formulated to have a density between anaverage density of a serum fraction of whole blood and a cell-containingfraction of whole blood, and to be flowable in whole blood.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawings in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1A is a side perspective view of a blood collection tube having apolymerizable separator substance that can harden.

FIG. 1B is a side perspective view of the blood collection of tube ofFIG. 1A after adding whole blood.

FIG. 1C is a side perspective view of the blood collection tube of FIG.1B after centrifugation.

DETAILED DESCRIPTION Collection Tube

In FIG. 1A blood collection tube 100 generally comprises tube 110, plug120, and separator substance 150, where tube 110 has lumen 115. Tube 110is preferably manufactured out of a suitably rigid material to support avacuum within lumen 115. Example materials include hard plastics, glass,or other similar materials. Lumen 115 is of sufficient volume to hold adesirable sample of whole blood or liquid. Typical volumes range from afew ml to 10 ml or greater. Plug 120 fits sufficiently snug into tube110 to maintain the vacuum within lumen 115. It is contemplated thatplug 120 is manufactured to provide a color code or other indicationthat separator substance 150 is disposed within lumen 115. An example ofan acceptable tube that can be used to produce collection tube 100includes the Vacutainer® specimen collection products developed byBecton, Dickenson and Company (Franklin Lakes, N.J. USA 07417).

Although the preferred embodiment includes tube 110, it is contemplatedthat collection tube 100 could be replaced with other vessels thatcontain a liquid and optionally support a vacuum. Alternative examplesof vessels include flasks, jars, beakers, bottles, or phials. Thecontemplated alternative vessels have utility when the inventive subjectmatter is applied to alternative markets beyond blood collection.

In a preferred embodiment, collection tube 100 is produced by disposingseparator substance 150 within lumen 115, and introducing a vacuumwithin lumen 115 in preparation for sale. It is also preferred that nomore than about 1 ml, or about 2 grams, of separator substance 150 isdisposed into lumen 115 for a typical 10 ml collection tube. It iscontemplated that other amounts, more or no more than 1 ml, could alsobe used to fit a specific use case. For example a smaller version oftube 110 would require less of separator substance 150, while a largerversion might require more to make an adequate sealed barrier.

The optional vacuum can be introduced by simply decompressing the volumeof lumen 115 by using a suitable pump. The term “vacuum” within thecontext of this document means a partial vacuum having a pressure lowerthan the pressure external to tube 110.

It is also contemplated that a user could add one or more separatorsubstances to a collection tube after purchase, as opposed to having aseparator substance pre-disposed within the tube.

FIG. 1B represents an exemplary embodiment of a blood collection tubeafter introduction of blood 140, and before centrifugation. Althoughblood 140 is shown on top of separator substance 150, the two might havecharacteristics in which they are free to flow or mix.

FIG. 1C represents an exemplary embodiment of a blood collection tubeafter centrifugation. During centrifugation blood 140 separates intoserum fraction 160 and cell-containing fraction 170. When separatorsubstance 150 has a density that is intermediate to that of serumfaction 160 and cell-containing fraction 170, it migrates between thetwo fractions during centrifugation, thereby isolating fractions 160 and170 from each other. Separator substance 150 can then be rapidlyhardened through polymerization when triggered by a suitable energysource.

Separator Substance

Preferably separator substance 150 rapidly hardens during finalpolymerization to a hardness that is resistant to penetration by apipette, to decanting, or even to freezing.

Hardness can be measured using any suitable hardness scale including oneof the Shore hardness scales. The Shore 00 hardness scale is used tomeasure soft substances including gels or foams. The Shore A hardnessscale is used to measure substances having an intermediate hardnessincluding rubbers. The Shore D hardness scale is used to measure hardersubstances including plastics. Although the preceding Shore hardnessscales are used for different various substances, the scales all overlapat the low end of their spectrums. Therefore, a value of 10 on the ShoreD scale is harder than a value of 10 on the Shore A scale which in turnis harder than a value of 10 on the Shore 00 scale. Separator substance150 is preferably formulated to harden to at least 1 on the Shore 00hardness scale. More preferred embodiments of separator substance 150harden further to at least 10 on the Shore A hardness scale. In yetother embodiments separator substance 150 harden even further to atleast 10 on the Shore D hardness scale.

Within the context of this document, the term “rapidly hardens” means toharden to at least 1 on the Shore 00 hardness scale within at least 10minutes. One of the aspects of the inventive subject matter isappreciating that a shorter time to harden can be advantageous over alonger timer. Having separator substance that hardens within a fewminutes, for example, could be important for a hospital to analyze asample in a critical life or death situation. In preferred embodiments,the time to harden is no more than 5 minutes, more preferably no morethan 1 minute, and most preferably no more than 10 seconds.

The hardened barrier of preferred separator substances adheres to thewalls of lumen 115 substantially sealing the cell-containing fractionand protecting the fractions from contamination due to diffusion,agitation, sample extraction, or other undesirable interaction. Inpreferred embodiments the final thickness of the barrier is no more than5 mm.

Separator substance 150 is preferably a biocompatible organic polymer.Among other things, biocompatibility means that the separator substance150 does not interfere with or alter any of the characteristics of thesubstances being tested. In the case of blood, for example, theseparator substance 150 should not interfere with pH, pigmentation, anyof the enzymes being tested, or with concentrations of proteins, gases,or any other components.

In yet other embodiments, it is contemplated that the substance couldinclude a component that intentionally reacts with the sample beingseparated. For example, the separator substance could include acoagulant, blood thinner, or other substance that interacts with wholeblood.

In blood separation tubes, the separator substance 150 should have adensity of between about 1.01-1.09 g/cm³, and most preferably about 1.04g/cm³. Unless the context dictates otherwise, all ranges herein are tobe interpreted as being inclusive of their endpoints.

An acceptable separator substance can include a polyester backbonesimilar to those described in U.S. Pat. Nos. 6,361,700 and 6,248,844,both of which are incorporated by reference herein. Polymerization ispreferably carried out to achieve the desired density of between about1.04-1.06 g/cm³. However, and in contrast to the methods andcompositions provided in the '700 and '844 patent, polymerization is notrun to completion but stopped using a polymerization terminator (e.g.,using radical quenchers, catalyst complexing agent, etc.) in a minimumamount effective to stop further polymerization.

As the sample contacts the incompletely cured polymer (separatorsubstance 150), it is contemplated that the polymerization terminator isdiluted to a concentration that allows the polymerization to bere-initiated. Prior to re-initiation, blood 140 is separated in thecontainer by centrifugation, which will leave cell-containing fraction170 in the bottom portion of tube 110 and serum fraction 160 in theupper portion of tube 110, wherein both fractions are separated by theincompletely cured polymer (separator substance 150). Re-initiation ofpolymerization may be assisted by irradiating the polymer with UV lightor other suitable energy source. Thus, it should be appreciated that thepolymeric is additionally cured after the separation is completed andthe so separated serum can then be accessed without contamination of apipette, decanted, or even frozen. Moreover, it should be recognizedthat the final cured barrier layer is substantially permanent (i.e.,stable over several days, or even weeks).

While it is generally acceptable that collection tube 100 include apolyester polymer as separator substance 150, it should be noted thatthe exact nature of the polymeric material is not limiting to theinventive subject matter, and that numerous alternative polymers arealso suitable. Indeed all known polymers suitable for whole bloodseparation are deemed appropriate for use herein, including silicon oil,polyamides, olefinic polymers, polyacrylates polyesters and copolymersthereof, polysilanes, and polyisoprenes. To achieve a desired initialdensity (typically between about 1.03 and 1.05), it is contemplated thatthe density may be adjusted by virtue of molecular composition, as wellas by inclusion of appropriate filler material (e.g., silica, latex, orother inert material). For example, suitable polymeric materials aredescribed in U.S. Pat. No. 3,647,070, 3,920,557, or 3,780,935, or in EP0 928 301 or 0 705 882, which are incorporated by reference herein.Furthermore, it is contemplated that the serum separators may includeadditional materials and/or reagents to achieve a desired bio-reactivepurpose. For example, the separators presented herein may include EDTA,heparin, citrate, dextrose, etc. It should be noted that the term“serum” is used herein to also include plasma, and other substantiallycell free fluids derived from whole blood.

Depending on the particular material, it is contemplated that the modeand/or mechanism of polymerization to the separator polymer may varyconsiderably, and all know manners of polymerization are deemed suitablefor use herein. For example, contemplated polymerizations includevarious radical or cationic polymerizations (e.g., using photolabilecompounds, radical startes, etc.), condensation polymerizations,esterifications, amide formation, etc. Thus, reactive groups willespecially include acid groups (and most preferably mono- anddicarboxylic groups), conjugated diene groups, aromatic vinyl groups,and alkyl(meth)acrylate. Such exemplary reactive groups and reactionconditions are described, for example, in U.S. Pat. No. 6,989,226, whichis incorporated by reference herein. It should furthermore beappreciated that the reactive groups can be coupled to the terminus of apolymer as end groups as described in WO 99/64931, which is incorporatedby reference herein, or that the reactive groups may be provided aspendant groups (e.g., as described in U.S. Pat. No. 5,336,736,incorporated by reference herein).

It is generally preferred that polymerization is fully supported byreactive groups on pre-polymer, but additional reagents may also besuitable, including radical starters, including those described in U.S.Pat. Nos. 5,582,954, 4,894,315, and 4,460,675, which are incorporated byreference herein. Additionally contemplated separator substances alsoinclude those that provide a crosslinking group to the polymer such thatthe polymer has reactive groups that react with a bifunctionalcrosslinker (e.g., ethylenically unsaturated compounds) to thereby formcrosslinked polymers. Yet additional contemplated separator substancesalso include those having promoters that accelerate polymerization.

An acceptable example of separator substance 150 includes a substanceknown as “M1L1A1” co-developed by the University of Maryland andUniversity of California Irvine. M1L1A1 is a polymeric separatorsubstance comprising the following: (M1) a monomer Trimethylolpropanepropoxylate triacrylate from Sigma-Aldrich Cat. No. 407577, (L1) CYTECAliphatic Urethane Acrylate EBECRYL 230 from Cytec Industries, Inc., and(A1) Additol BDK, 2,2-Dimethoxy-1,2-diphenyl-ethan-1-one also from CytecIndustries, Inc. Additionally, M1L1A1 has desirable properties for usewith whole blood including an adjustable density by adding fumed silica,it is flowable in whole blood when centrifuged, thixotropic, a hardnessgreater than 10 on the Shore A hardness scale after polymerization,hardens in no more than 10 seconds under exposure to UV light,biocompatible with whole blood, and forms a hardened seal impermeable tothe cell-containing fraction of whole blood and that is resistant topenetration of a pipette. M1L1A1 hardens under a UV light source thatradiates light in the wavelengths from 10 nm to 450 nm. A preferred UVlight source radiates in the range 250 nm to 400 nm. All suitable energysources are contemplated for triggering polymerization. It iscontemplated that an existing centrifuge having a UV source can be usedto polymerize the separator substance, or a centrifuge can incorporate asuitable energy source capable of triggering polymerization.

Preferably, the temperature of the collection tube contents changes byno more than 10 degrees Celsius during polymerization; more preferablyby no more than 5 degrees Celsius. Short exposure times ensures thesample will maintain appropriate pigmentation levels, gas levels,temperatures, protein levels, or other characteristics associated withwhole blood.

Alternative Embodiments

Although the preferred embodiment of the inventive subject matterprimarily focuses on blood collection tubes, one should recognize thatthe systems, apparatus, and methods presented herein can be applied toalternative markets beyond blood collection tubes. Similar techniques tothose disclosed herein can also be employed to separate nearly any fluidhaving more than one constituent phase. For example, a separatorsubstance can be provided to separate fluids including urine, watersamples, oil, wine, or other multi-phase fluids. For fluids having morethan two phases, it is contemplated that a collection tube can containmore than one separator substances which are used to separate at leastthree phases of the fluid.

In some applications, a user of a collection tube might require accessto the cell containing portion as well as a serum containing portion. Insuch cases the separator substance can be tuned to have an appropriatelower hardness (no more than about 10 on the Shore 00 hardness scale) toallow decanting the serum portion without disturbing the cell-containingportion. A pipette can then penetrate the separator substance barrier toaccess the cell-containing portion. Alternatively, the collection tubecan be produced to have two opens ends where the “top” and “bottom” ofthe tube both have plugs. The cell-containing portion can then beaccessed through the bottom plug. The bottom plug can be sized anddimensioned to have a rounded shape to fit the shape of a centrifugeholder and to provide support during centrifugation.

Advantages

There are numerous advantages of producing a collection tube having arapidly hardening separator substance for use in whole blood separation.One advantage includes that the fractions of whole blood are effectivelyisolated from each other permanently with little or no chance ofcontamination due to diffusion, agitation, sample extraction, or otherundesired interaction. In addition, using a separator substance thatsubstantially hardens as a separation layer implies one requires asmaller amount of material than when using a thixotropic gel as aseparator substance, for example. Consequently, less time is requiredfor centrifugation to achieve full separation because there is lessmaterial to migrate to the separation layer and more volume within thetube is available for a blood sample.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

1. A method of separating blood comprising: providing access to a bloodcollecting tube containing a separator substance that polymerizes within10 minutes to at least 1 on the Shore 00 hardness scale when triggeredby a suitable energy source; disposing whole blood into the bloodcollection tube; separating the whole blood into a serum fraction and acell containing fraction separated by the separator substance; andexposing the separator substance after separation to an energy sourcetriggering polymerization of the separator substance within 10 minutesto at least 1 on the Shore 00 hardness scale.
 2. The method of claim 1,wherein the step of separating the whole blood comprises centrifugingthe blood collection tube containing the whole blood.
 3. The method ofclaim 1, wherein the separator substance comprises sufficient reactivegroups to polymerize within 10 minutes.
 4. The method of claim 1,wherein the separator substance comprises a sufficient amount of atleast one promoter to polymerize in 10 minutes.
 5. The method of claim1, wherein the separator substance has a cured hardness greater than 10on the Shore A hardness scale.
 6. The method of claim 5, wherein theseparator substance has a cured hardness greater than 10 on the Shore Dhardness scale.
 7. The method of claim 1, wherein the separatorsubstance hardens within 5 minutes to at least 1 on the Shore 00hardness scale.
 8. The method of claim 7, wherein the separatorsubstance hardens within 5 minutes to at least 10 on the Shore Ahardness scale.
 9. The method of claim 1, wherein the separatorsubstance is formulated to have a density between an average density ofa serum fraction of whole blood and a cell-containing fraction of wholeblood, and that is further formulated to be flowable in whole blood. 10.The method of claim 9, wherein the separator substance afterpolymerization is impermeable to the cell-containing fraction of wholeblood.
 11. The method of claim 1, wherein the separator substance isbiocompatible with whole blood.
 12. The method of claim 1, wherein theenergy source comprises UV light.
 13. The method of claim 1, wherein thestep of exposing the separator substance includes exposing the substanceto UV light having wavelengths in a range from 10 nm to 450 nm.
 14. Themethod of claim 13, wherein the step of exposing the separator substanceincludes exposing the substance to UV light having wavelengths in arange from 250 nm to 400 nm.
 15. The method of claim 1, wherein the stepof exposing the separator substance includes using the energy sourceincorporated into a centrifuge.
 16. The method of claim 1, wherein thetube changes temperature by no more than 10 degrees Celsius within the10 minutes during polymerization.
 17. The method of claim 1, wherein theamount of the separator substance disposed within the lumen results in abarrier thickness of no more than 5 mm after polymerization.